The life cycle of flowering plants in general can be divided into three growth phases: vegetative, inflorescence, and floral (Poethig 1990). In the vegetative phase, the shoot apical meristem (SAM) generates leaves that will later ensure the resources necessary to produce fertile offspring. Upon receiving the appropriate environmental and developmental signals, the plant switches to floral, or reproductive, growth and the SAM enters the inflorescence phase (I1) and gives rise to an inflorescence with flower primordia. During this phase, the fate of the SAM and the secondary shoots that arise in the axils of the leaves is determined by a set of meristem identity genes, some of which prevent and some of which promote the development of floral meristems. Two basic types of inflorescences have been identified in plants: determinate and indeterminate (Weberling, 1989). In determinate species, such as ryegrass, the SAM eventually produces floral organs and the production of meristems is terminated with a flower. The SAM of indeterminate species is not converted to a floral identity and will therefore only produce floral meristems from its periphery, resulting in a continuous growth pattern.
The regulation of meristem identity and floral transition has been investigated in a number of dicotyledonous plants including Arabidopsis, Antirrhinum, tomato, and tobacco. However; in agronomically important seed crops such as wheat, barley, rice, forage grasses, and other monocotyledonous plants, information on how floral transition is controlled is still limited. The present inventors have undertaken a molecular investigation of the regulation of meristem identity and the control of floral transition in perennial ryegrass Lolium perenne), a cool-season perennial forage grass native to Europe, temperate Asia, and North Africa.
There are several reasons for such an investigation. Firstly the production of culm (stem) and seed head (inflorescence) formation decreases the feeding value of forage grasses. The leaf blades are more digestible, richer in crude protein and poorer in cell-wall constituents than sheaths and culms (Deinum and Dirvan, 1975; Wilman et al., 1976). The ageing of grasses is associated with an increase In lignification and a decrease in digestibility, which is markedly higher for the stems than for the leaves (Delagarde et al., 2000). Feeding trials on cows have documented that increasing the digestibility of forage grass leads to a daily increase in feed uptake and milk production (Oba and Allen, 1999). Secondly, maintenance of a vegetative forage grass requires a frequent mechanical defoliation system, which is both costly and time consuming. Too intensive defoliation can severely decrease the photosynthetic capacity of the plant and in the worst case destroy the regeneration capacity. Thirdly, flowering in many plants is associated with an uncontrollable gene flow from cultivated to wild species via the active spread of pollen. Fourthly, flowering in many perennial plants is also associated with an exposure of grass pollen allergens. A grass cultivar with an extended vegetative growth associated with decreased or even eliminated inflorescence production would thus be agronomically attractive.
In terms of plant development, the aerial parts of ryegrass are produced by the apex positioned on the base crown a few millimeters above the ground and surrounded by developing leaves (FIG. 1A). During vegetative growth the apical meristem generates lateral meristems initially recognised as semicircular ridges along the main axis. These become the leaf primordia. This morphological pattern does not change until the apex has been induced to flower by elevated temperatures and increasing day length. Flowering in perennial ryegrass is induced by a vernalization period of 12 to 14 weeks below 5° C. followed by secondary induction with long-day photoperiods (generally, more daylight hours than dark hours and, in particular, LD, 16 h of light, 8 h of darkness) and temperatures above 15 to 20° C. Upon transition to reproductive growth, the apical meristem and later also the lateral meristems start to expand and eventually turn into groups of inflorescences (spikelets), each containing three to 10 floral meristems. The flowers of the ryegrass inflorescence are arranged in a cymose, always terminating apical growth with the production of a terminal flower. In this way ryegrass represents a determinate plant architecture also seen and described at the molecular level in dicot plants such as tobacco (Amaya et al., 1999) and tomato (Pnueli et al., 1998). In contrast to plants such as Arabidopsis and Antirrhinum, ryegrass has a determinate (cymose) inflorescence. The TERMINAL FLOWER 1 (TFL1) gene of Arabidopsis and its homolog CENTRORADIALIS (CEN) in Antirrhinum have been identified as a group of genes that specify an indeterminate identity of inflorescence meristems. Mutations in TFL1/CEN result in the conversion of the inflorescence into a terminal flower (Shannon and Meeks-Wagner, 1991; Alvarez et al., 1992;). In addition to its effect on meristem fate, TFL1 also extends the vegetative phase of Arabidopsis (Shanon and Meeks-Wagner, 1991, Ratcliff et al., 1998), but CEN does not seem to have a flowering time role in Antirrhinum (Bradley et al., 1996). CEN and TFL1 proteins have sequence similarity with mammalian phosphatidylethanolamine-binding proteins (PEBPs). The FLOWERING LOCUS T (FT) gene also belongs to the family of plant PEBP genes, but has been shown to play an opposite role to TFL1 in mediating flower inducing signals in Arabidopsis (Kardailsky et al., 1999; Kobayashi et al., 1999). Therefore the family of plant PEBP genes comprise a number of homologues proteins with different properties in relation to floral control. These differences are further revealed by the expression of different plant PEBPs from a constitutive promoter in different plant species. Expression of the TFL1 gene in tobacco from the 35S CaMV constitutive promoter, for example, does not affect the flowering time and does not affect the plant architecture of tobacco (Amaya et al., 1999).
The differences in gene function are also reflected in the different expression patterns by plant PEBPs. In Arabidopsis and Antirrhinum, TFL1/CEN is expressed in the centre of the SAM. Upon transition from vegetative to reproductive growth, the expression of these genes increases (Bradley et al., 1996, 1997). Expression of the floral meristem identity genes such as LFY, AP1 and CAL is also increased in the SAM upon transition to reproductive growth, but the expression is confined to the developing flowers (Mandel et al., 1992; Bradley et al., 1997; Ratcliffe et al., 1999). In tobacco the CET2/CET4 genes are mainly expressed in the axillary meristems and not in the SAM (Amaya et al., 1999).